Assessment of active methanogenic archaea in a methanol-fed upflow anaerobic sludge blanket reactor.

Methanogenic archaea enrichment of a granular sludge was undertaken in an upflow anaerobic sludge blanket (UASB) reactor fed with methanol in order to enrich methylotrophic and hydrogenotrophic methanogenic populations. A microbial community assessment, in terms of microbial composition and activity-throughout the different stages of the feeding process with methanol and acetate-was performed using specific methanogenic activity (SMA) assays, quantitative real-time polymerase chain reaction (qPCR), and high-throughput sequencing of 16S ribosomal RNA (rRNA) genes from DNA and complementary DNA (cDNA). Distinct methanogenic enrichment was revealed by qPCR of mcrA gene in the methanol-fed community, being two orders of magnitude higher with respect to the initial inoculum, achieving a final mcrA/16S rRNA ratio of 0.25. High-throughput sequencing analysis revealed that the resulting methanogenic population was mainly composed by methylotrophic archaea (Methanomethylovorans and Methanolobus genus), being also highly active according to the RNA-based assessment. SMA confirmed that the methylotrophic pathway, with a direct conversion of methanol to CH4, was the main step of methanol degradation in the UASB. The biomass from the UASB, enriched in methanogenic archaea, may bear great potential as additional inoculum for bioreactors to carry out biogas production and other related processes.

Role of Thiobacillus thioparus in the biodegradation of carbon disulfide in a biofilter packed with a recycled organic pelletized material.

This study reports the biodegradation of carbon disulfide (CS2) in air biofilters packed with a pelletized mixture of composted manure and sawdust. Experiments were carried out in two lab-scale (1.2 L) biofiltration units. Biofilter B was seeded with activated sludge enriched previously on CS2-degrading biomass under batch conditions, while biofilter A was left as a negative inoculation control. This inoculum was characterized by an acidic pH and sulfate accumulation, and contained Achromobacter xylosoxidans as the main putative CS2 biodegrading bacterium. Biofilter operation start-up was unsuccessfully attempted under xerophilic conditions and significant CS2 elimination was only achieved in biofilter A upon the implementation of an intermittent irrigation regime. Sustained removal efficiencies of 90-100 % at an inlet load of up to 12 g CS2 m(-3) h(-1) were reached. The CS2 removal in this biofilter was linked to the presence of the chemolithoautotrophic bacterium Thiobacillus thioparus, known among the relatively small number of species with a reported capacity of growing on CS2 as the sole energy source. DGGE molecular profiles confirmed that this microbe had become dominant in biofilter A while it was not detected in samples from biofilter B. Conventional biofilters packed with inexpensive organic materials are suited for the treatment of low-strength CS2 polluted gases (IL <12 g CS2 m(-3) h(-1)), provided that the development of the adequate microorganisms is favored, either upon enrichment or by inoculation. The importance of applying culture-independent techniques for microbial community analysis as a diagnostic tool in the biofiltration of recalcitrant compounds has been highlighted.

The Metallotolerance and Biosorption of As(V) and Cr(VI) by Black Fungi.

A collection of 34 melanized fungi isolated previously from anthropogenic contaminated sites were assessed for their tolerance to toxic concentrations of As(V) and Cr(VI) anions. Three strains of the species Cyphellophora olivacea, Rhinocladiella similis, and Exophiala mesophila (Chaetothyriales) were identified as hyper-metallotolerant, with estimated IC50 values that ranged from 11.2 to 16.9 g L-1 for As(V) and from 2.0 to 3.4 g L-1 for Cr(VI). E. mesophila and R. similis were selected for subsequent assays on their biosorption capacity and kinetics under different pH values (4.0 and 6.5) and types of biomass (active and dead cells and melanin extracts). The fungal biosorption of As(V) was relatively ineffective, but significant removal of Cr(VI) was observed from liquid cultures. The Langmuir model with second-order kinetics showed maximum sorption capacities of 39.81 mg Cr6+ g-1 for R. similis and 95.26 mg Cr6+ g-1 for E. mesophila on a dry matter basis, respectively, while the kinetic constant for these two fungi was 1.32 × 10-6 and 1.39 × 10-7 g (mg Cr6+ min)-1. Similar experiments with melanin extracts of E. mesophila showed maximum sorption capacities of 544.84 mg Cr6+ g-1 and a kinetic constant of 1.67 × 10-6 g (mg Cr6+ min)-1. These results were compared to bibliographic data, suggesting that metallotolerance in black fungi might be the result of an outer cell-wall barrier to reduce the diffusion of toxic metals into the cytoplasm, as well as the inner cell wall biosorption of leaked metals by melanin.

Characterization of melanin from Exophiala mesophila with the prospect of potential biotechnological applications.

Introducion: Fungal melanin is an underexplored natural biomaterial of great biotechnological interest in different areas. This study investigated the physical, chemical, electrochemical, and metal-binding properties of melanin extracted from the metallotolerant black fungus Exophiala mesophila strain IRTA-M2-F10. Materials and methods: Specific inhibitory studies with tricyclazole and biochemical profiling of whole cells by synchrotron radiation-based Fourier-transform infrared spectral microscopy (SR-FTIRM) were performed. An optimized extraction protocol was implemented, and purified fungal melanin was characterized using an array of spectrophotometric techniques (UV-Vis, FTIR, and EPR) and by cyclic voltammetry (CV) experiments. The metal-binding capacity of melanin extracts was also assessed by using Cr(VI) as a model heavy metal. Results: Inhibitory studies indicated that 1,8-dihydroxynaphthalene may be the main precursor molecule of E. mesophila melanin (DHN-melanin). The biochemical characterization of fungal melanin extracts were benchmarked against those from two melanins comprising the precursor molecule L-3,4-dihydroxiphenylalanine (DOPA-melanin): extracts from the ink of the cephalopod Sepia officinalis and DOPA-melanin synthesized in the laboratory. The CV results of melanin extracts incubated with and without cell suspensions of the electroconductive bacterium Geobacter sulfurreducens were indicative of novel semiquinone/hydroquinone redox transformations specific for each melanin type. These interactions may play an important role in cation exchange for the adsorption of metals and in microbial interspecies electron transfer processes. Discussion: The obtained results provided further evidence for the DHN-nature of E. mesophila melanin. The FTIR profiling of melanin extracts exposed to Cr(VI), compared to unexposed melanin, resulted in useful information on the distinct surface-binding properties of fungal melanin. The parameters of the Langmuir and Freundlicht isotherms for the adsorption of Cr(VI) were determined and compared to bibliographic data. Altogether, the inherent properties of fungal melanin suggest its promising potential as a biomaterial for environmental applications.

Use of isotopic (C, Cl) and molecular biology tools to assess biodegradation in a source area of chlorinated ethenes after biostimulation with Emulsified Vegetable Oil (EVO).

Enhanced In Situ Bioremediation (EISB) using Emulsified Vegetable Oil (EVO) as a long-term electron donor has gained prominence for the treatment of groundwater contaminated with chlorinated ethenes (CEs). This study explores the potential of isotopic and molecular biology tools (MBT) to investigate the CEs (PCE, TCE and cis-DCE) bioremediation using EVO in a contaminated site. A multiple approach using C and Cl-CSIA, quantification of Dehalococcoides (Dhc) and specific reductive dechlorination (RD) gene population, and hydrochemical data in microcosm experiments and field samples was applied. Despite the high partitioning of CEs into the EVO phase, the carbon isotopic values of the remaining CEs fraction in the aqueous phase did not exhibit significant changes caused by phase partitioning in laboratory experiments. Both microcosm experiments and field data revealed a rapid RD of PCE and TCE, resulting in the transient accumulation of cis-DCE, which was slowly degraded to vinyl chloride (VC). These results agreed with the presence of Dhc populations and a shift to stronger reducing conditions in the field: i) RD functional genes (tceA, vcrA and bvcA) exhibited a trend to higher values and ii) a substantial increase in Dhc populations (up to 30% of the total bacterial populations) was observed over time. The dual-element isotope slope ΛC-Cl for RD of cis-DCE obtained from field data (ΛC - Cl = 5 ± 3) was similar to the one determined from the microcosm experiments under controlled anoxic conditions (ΛC - Cl = 4.9 ± 0.8). However, ΛC-Cl values differ from those reported so far for laboratory studies with Dhc strains and mixed cultures containing Dhc, i.e., between 8.3 and 17.8. This observation underscores the potential variety of reductive dehalogenases involved during cis-DCE RD and the importance of determining site-specific Λ and ɛ values in order to improve the identification and quantification of transformation processes in the field.

Use of wood and cork in biofilters for the simultaneous removal of nitrates and pesticides from groundwater.

About 13% and 7% of monitored groundwater stations in Europe exceed the permitted levels of nitrates (50 mg NO3- L-1) or pesticides (0.1 µg L-1), respectively. Although slow sand filtration can remove nitrates via denitrification when oxygen is limited, it requires an organic carbon source. The present study evaluates the performance of the use of wood pellets and granulated cork as carbon sources in bench-scale biofilters operated under water-saturated and water-unsaturated conditions for more than 400 days. The biofilters were monitored for nitrate (200 mg L-1) and pesticide (mecoprop, diuron, atrazine, and bromacil, each at a concentration of 5 µg L-1) attenuation, as well as for the formation of nitrite and pesticide transformation products. Microbiological characterization of each biofilter was also performed. The water-saturated wood biofilter achieved the best nitrate removal (>99%), while the cork biofilters lost all denitrification power over time (from 38% to no removal). The unsaturated biofilter columns were not effective for removing nitrates (20-30% removal). As for pesticides, all the biofilters achieved high removal rates of mecoprop and diuron (>99% and >75%, respectively). Atrazine removal was better in the wood-pellet biofilters than the cork ones (68-96% vs. 31-38%). Bromacil was only removed in the water-unsaturated cork biofilter (67%). However, a bromacil transformation product was formed there. The water-saturated wood biofilter contained the highest number of denitrifying microorganisms, with Methyloversatilis as the characteristic genus. Microbial composition could explain the high removal of pesticides and nitrates achieved in the wood-pellet biofilter. Overall, the results indicate that wood-pellet biofilters operated under water-saturated conditions are a good solution for treating groundwater contaminated with nitrates and pesticides.

Modulation of gut microbiota and intestinal immune response in gilthead seabream (Sparus aurata) by dietary bile salt supplementation.

Given their role in lipid digestion, feed supplementation with bile salts could be an economic and sustainable solution to alterations in adiposity and intestinal inflammation generated by some strategies currently used in aquaculture. An important part of the metabolism of bile salts takes place in the intestine, where the microbiota transforms them into more toxic forms. Consequently, we aimed to evaluate the gut immune response and microbial populations in gilthead seabream (Sparus aurata) fed a diet supplemented with a blend of bile salts with proven background as a regulator of lipid metabolism and fat content. After the 90-day feeding trial, a differential modulation of the microbiota between the anterior and posterior intestine was observed. While in the anterior intestine the relative abundance of Desulfobacterota doubled, in the posterior intestine, the levels of Firmicutes increased and Proteobacteria, Actinobacteriota, and Campylobacterota were reduced when supplementing the diet with bile salts. Even so, only in the anterior intestine, there was a decrease in estimated richness (Chao1 and ACE indices) in presence of dietary bile salts. No significant differences were displayed in alpha (Shannon and Simpson indices) nor beta-diversity, showing that bile sales did not have a great impact on the intestinal microbiota. Regarding the gene expression profile in 2 h postprandial-fish, several changes were observed in the analyzed biomarkers of epithelial integrity, nutrient transport, mucus production, interleukins, cell markers, immunoglobulin production and pathogen recognition receptors. These results may indicate the development of an intestinal immune-protective status to tackle future threats. This work also suggests that this immune response is not only regulated by the presence of the dietary bile salts in the intestine, but also by the microbial populations that are in turn modulated by bile salts. After a fasting period of 2 days, the overall gene expression profile was stabilized with respect to fish fed the unsupplemented diet, indicating that the effect of bile salts was transient after short periods of fasting. On the balance, bile salts can be used as a dietary supplement to enhance S. aurata farming and production without compromising their intestinal health.

The potential of a combination of pungent spices as a novel supplement in gilthead seabream (Sparus aurata) diets to aid in the strategic use of fish oil in aquafeeds: a holistic perspective.

This work studied the potential of a combination of pungent spices (capsicum, black pepper, ginger, and cinnamaldehyde) to be used as a supplement in diets of gilthead seabream (Sparus aurata; 44.1 ± 4.2 g). During 90 days, fish were fed three experimental diets with low inclusion of fish oil and containing poultry fat as the main source of lipids, supplemented with graded levels of the tested supplement: 0 (control), 0.1 (SPICY0.1%), and 0.15% (SPICY0.15%). As a result, the pungent spices enhanced the growth performance, the activity of the bile-salt-activated lipase in the intestine, and decreased fat deposit levels within enterocytes. The SPICY0.1% diet reduced the feed conversion ratio and the perivisceral fat index and lipid deposits in the liver. Moreover, the ratio of docosahexaenoic acid/eicosapentaenoic acid in fillet increased in fish fed the SPICY0.1% diet, while the hepatic levels of docosahexaenoic acid and total n-3 polyunsaturated fatty acids increased in fish fed the SPICY0.15% diet. Furthermore, there was an effect on the expression of some biomarkers related to lipid metabolism in 2-h postprandial fish (fasn, elovl6, scd1b, cyp7a1, lpl, and pparß), and in 48 h fasted-fish fed with the SPICY0.1% diet, a regulation of the intestinal immune response was indicated. However, no significant differences were found in lipid apparent digestibility and proximate macronutrient composition. The spices did not affect biomarkers of hepatic or oxidative stress. No differences in microbial diversity were found, except for an increase in Simpson's Index in the posterior intestine of fish fed the SPICY0.1% diet, reflected in the increased relative abundance of the phylum Chloroflexi and lower relative abundances of the genera Campylobacter, Corynebacterium, and Peptoniphilus. In conclusion, the supplementation of gilthead seabream diets with pungent spices at an inclusion of 0.1% was beneficial to enhance growth performance and feed utilization; reduce fat accumulation in the visceral cavity, liver, and intestine; and improve the fish health status and condition. Results suggest that the tested supplement can be used as part of a nutritional strategy to promote a more judicious use of fish oil in fish diets due to its decreasing availability and rising costs.

The use of recovered struvite and ammonium nitrate in fertigation in a horticultural rotation: agronomic and microbiological assessment.

Phosphorus and nitrogen recovery from wastewater as struvite and ammonium nitrate (AN) may be viable alternative fertilizers to boost circularity in horticulture. A 2-year fertigated crop rotation in soil under greenhouse conditions was evaluated to determine the efficiency of both recovered products as raw materials for a nutrient solution (NS) manufacture. The effects of these treatments versus synthetic fertilizers were compared in terms of crop performance, plant nutrient uptake, soil chemistry and microbiota. This is the first study to implement struvite through fertigation as the sole source of P in soil crops. Results showed that both recovered products can be used as fertilizers in NS, due to the similar response to the control for different parameters and crops (tomato, lettuce, and cauliflower). However, the AN treatment showed lower yield in the first tomato crop, which results may depend on the cultivar ammonium tolerance. Besides, the concentration of heavy metals in fruits/leaves was below the permissible limits. Total and Olsen phosphorus soil analysis revealed no differences among treatments, resulting in a similar performance of P-struvite to commercial phosphate. Bulk soil bacteria structure, richness and relative dominance were increased over time, while archaea only showed lower evenness, both despite the fertilization strategy. Shannon diversity was not significantly affected. A predominance of ammonia-oxidizing bacteria (AOB) versus archaea (AOA) was observed, while nitrite-oxidizing bacteria (NOB), dominated by Nitrospira, increased with fertigation. Our results demonstrate that fertilizer blends for NS containing recovered nutrients are a feasible alternative to synthetic fertilizers.

Assessment of a novel microalgae-cork based technology for removing antibiotics, pesticides and nitrates from groundwater.

Groundwater pollution has increased in recent years due to the intensification of agricultural and livestock activities. This results in a significant reduction in available freshwater resources. Here, we have studied the long term assessment of a green technology (1-4 L/day) based on a photobioreactor (PBR) containing immobilised microalgae-bacteria in polyurethane foam (PF) followed by a cork filter (CF) for removing nitrates, pesticides (atrazine and bromacil), and antibiotics (sulfamethoxazole and sulfacetamide) from groundwater. The prototype was moderately effective for removing nitrates (58%) at an HRT of 8 days, while its efficiency decreased at a HRT of 4 and 2 days (<20% removal). The combined use of PBR-CF enabled antibiotics and pesticides to be attenuated by up to 95% at an HRT of 8 days, but their attenuation decreased with shorter HRT, with pesticides being the compounds most affected (reducing from 97 to 98% at an HRT of 8 days to 23-45% at an HRT of 2 days). Pesticide transformation products were identified after the CF, supporting biodegradation as the main attenuation process. A gene-based metataxonomic assessment linked the attenuation of micropollutants to the presence of specific pesticide biodegradation species (e.g. genus Phenylobacterium, Sphingomonadaceae, and Caulobacteraceae). Therefore, the results highlighted the potential use of microalgae and cork to treat polluted groundwater.

Polyphasic approach for assessing changes in an autochthonous marine bacterial community in the presence of Prestige fuel oil and its biodegradation potential.

A laboratory experiment was conducted to identify key hydrocarbon degraders from a marine oil spill sample (Prestige fuel oil), to ascertain their role in the degradation of different hydrocarbons, and to assess their biodegradation potential for this complex heavy oil. After a 17-month enrichment in weathered fuel, the bacterial community, initially consisting mainly of Methylophaga species, underwent a major selective pressure in favor of obligate hydrocarbonoclastic microorganisms, such as Alcanivorax and Marinobacter spp. and other hydrocarbon-degrading taxa (Thalassospira and Alcaligenes), and showed strong biodegradation potential. This ranged from >99% for all low- and medium-molecular-weight alkanes (C(15)-C(27)) and polycyclic aromatic hydrocarbons (C(0)- to C(2)- naphthalene, anthracene, phenanthrene, dibenzothiophene, and carbazole), to 75-98% for higher molecular-weight alkanes (C(28)-C(40)) and to 55-80% for the C(3) derivatives of tricyclic and tetracyclic polycyclic aromatic hydrocarbons (PAHs) (e.g., C(3)-chrysenes), in 60 days. The numbers of total heterotrophs and of n-alkane-, aliphatic-, and PAH degraders, as well as the structures of these populations, were monitored throughout the biodegradation process. The salinity of the counting medium affects the counts of PAH degraders, while the carbon source (n-hexadecane vs. a mixture of aliphatic hydrocarbons) is a key factor when counting aliphatic degraders. These limitations notwithstanding, some bacterial genera associated with hydrocarbon degradation (mainly belonging to α- and γ-Proteobacteria, including the hydrocarbonoclastic Alcanivorax and Marinobacter) were identified. We conclude that Thalassospira and Roseobacter contribute to the degradation of aliphatic hydrocarbons, whereas Mesorhizobium and Muricauda participate in the degradation of PAHs.

Degradation of 4-n-nonylphenol under nitrate reducing conditions.

Nonylphenol (NP) is an endocrine disruptor present as a pollutant in river sediment. Biodegradation of NP can reduce its toxicological risk. As sediments are mainly anaerobic, degradation of linear (4-n-NP) and branched nonylphenol (tNP) was studied under methanogenic, sulphate reducing and denitrifying conditions in NP polluted river sediment. Anaerobic bioconversion was observed only for linear NP under denitrifying conditions. The microbial population involved herein was further studied by enrichment and molecular characterization. The largest change in diversity was observed between the enrichments of the third and fourth generation, and further enrichment did not affect the diversity. This implies that different microorganisms are involved in the degradation of 4-n-NP in the sediment. The major degrading bacteria were most closely related to denitrifying hexadecane degraders and linear alkyl benzene sulphonate (LAS) degraders. The molecular structures of alkanes and LAS are similar to the linear chain of 4-n-NP, this might indicate that the biodegradation of linear NP under denitrifying conditions starts at the nonyl chain. Initiation of anaerobic NP degradation was further tested using phenol as a structure analogue. Phenol was chosen instead of an aliphatic analogue, because phenol is the common structure present in all NP isomers while the structure of the aliphatic chain differs per isomer. Phenol was degraded in all cases, but did not affect the linear NP degradation under denitrifying conditions and did not initiate the degradation of tNP and linear NP under the other tested conditions.

Bioaugmentation of Native Fungi, an Efficient Strategy for the Bioremediation of an Aged Industrially Polluted Soil With Heavy Hydrocarbons.

The concurrence of structurally complex petroleum-associated contaminants at relatively high concentrations, with diverse climatic conditions and textural soil characteristics, hinders conventional bioremediation processes. Recalcitrant compounds such as high molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs) and heavy alkanes commonly remain after standard soil bioremediation at concentrations above regulatory limits. The present study assessed the potential of native fungal bioaugmentation as a strategy to promote the bioremediation of an aged industrially polluted soil enriched with heavy hydrocarbon fractions. Microcosms assays were performed by means of biostimulation and bioaugmentation, by inoculating a defined consortium of six potentially hydrocarbonoclastic fungi belonging to the genera Penicillium, Ulocladium, Aspergillus, and Fusarium, which were isolated previously from the polluted soil. The biodegradation performance of fungal bioaugmentation was compared with soil biostimulation (water and nutrient addition) and with untreated soil as a control. Fungal bioaugmentation resulted in a higher biodegradation of total petroleum hydrocarbons (TPH) and of HMW-PAHs than with biostimulation. TPH (C14-C35) decreased by a 39.90 ± 1.99% in bioaugmented microcosms vs. a 24.17 ± 1.31% in biostimulated microcosms. As for the effect of fungal bioaugmentation on HMW-PAHs, the 5-ringed benzo(a)fluoranthene and benzo(a)pyrene were reduced by a 36% and 46%, respectively, while the 6-ringed benzoperylene decreased by a 28%, after 120 days of treatment. Biostimulated microcosm exhibited a significantly lower reduction of 5- and 6-ringed PAHs (8% and 5% respectively). Higher TPH and HMW-PAHs biodegradation levels in bioaugmented microcosms were also associated to a significant decrease in acute ecotoxicity (EC50) by Vibrio fischeri bioluminiscence inhibition assays. Molecular profiling and counting of viable hydrocarbon-degrading bacteria from soil microcosms revealed that fungal bioaugmentation promoted the growth of autochthonous active hydrocarbon-degrading bacteria. The implementation of such an approach to enhance hydrocarbon biodegradation should be considered as a novel bioremediation strategy for the treatment of the most recalcitrant and highly genotoxic hydrocarbons in aged industrially polluted soils.

Aerobic nonylphenol degradation and nitro-nonylphenol formation by microbial cultures from sediments.

Nonylphenol (NP) is an estrogenic pollutant which is widely present in the aquatic environment. Biodegradation of NP can reduce the toxicological risk. In this study, aerobic biodegradation of NP in river sediment was investigated. The sediment used for the microcosm experiments was aged polluted with NP. The biodegradation of NP in the sediment occurred within 8 days with a lag phase of 2 days at 30 degrees C. During the biodegradation, nitro-nonylphenol metabolites were formed, which were further degraded to unknown compounds. The attached nitro-group originated from the ammonium in the medium. Five subsequent transfers were performed from original sediment and yielded a final stable population. In this NP-degrading culture, the microorganisms possibly involved in the biotransformation of NP to nitro-nonylphenol were related to ammonium-oxidizing bacteria. Besides the degradation of NP via nitro-nonylphenol, bacteria related to phenol-degrading species, which degrade phenol via ring cleavage, are abundantly present.

Chemical and microbial community analysis during aerobic biostimulation assays of non-sulfonated alkyl-benzene-contaminated groundwater.

A chemical and microbial characterization of lab-scale biostimulation assays with groundwater samples taken from an industrial site in which the aquifer had been contaminated by linear non-sulfonate alkyl benzenes (LABs) was carried out for further field-scale bioremediation purposes. Two lab-scale biodegradability assays were performed, one with a previously obtained gas-oil-degrading consortium and another with the native groundwater flora. Results for the characterization of the groundwater microbial population of the site revealed the presence of an important LAB-degrading microbial population with a strong degrading capacity. Among the microorganisms identified at the site, the detection of Parvibaculum lavamentivorans, which have been described in other studies as alkyl benzene sulfonates degraders, is worth mentioning. Incubation of P. lavamentivorans DSMZ13023 with LABs as reported in this study shows for the first time the metabolic capacity of this strain to degrade such compounds. Results from the biodegradation assays in this study showed that the indigenous microbial population had a higher degrading capacity than the gas-oil-degrading consortium, indicating the strong ability of the native community to adapt to the presence of LABs. The addition of inorganic nutrients significantly improved the aerobic biodegradation rate, achieving levels of biodegradation close to 90%. The results of this study show the potential effectiveness of oxygen and nutrients as in situ biostimulation agents as well as the existence of a complex microbial community that encompasses well-known hydrocarbon- and LAS-degrading microbial populations in the aquifer studied.

Breeding farm, level of feeding and presence of antibiotics in the feed influence rabbit cecal microbiota.

BACKGROUND: The effect of the production environment and different management practices in rabbit cecal microbiota remains poorly understood. While previous studies have proved the impact of the age or the feed composition, research in the breeding farm and other animal management aspects, such as the presence of antibiotics in the feed or the level of feeding, is still needed. Characterization of microbial diversity and composition of growing rabbits raised under different conditions could help better understand the role these practices play in cecal microbial communities and how it may result in different animal performance. RESULTS: Four hundred twenty-five meat rabbits raised in two different facilities, fed under two feeding regimes (ad libitum or restricted) with feed supplemented or free of antibiotics, were selected for this study. A 16S rRNA gene-based assessment through the MiSeq Illumina sequencing platform was performed on cecal samples collected from these individuals at slaughter. Different univariate and multivariate approaches were conducted to unravel the influence of the different factors on microbial alpha diversity and composition at phylum, genus and OTU taxonomic levels. The animals raised in the facility harboring the most stable environmental conditions had greater, and less variable, microbial richness and diversity. Bootstrap univariate analyses of variance and sparse partial least squares-discriminant analyses endorsed that farm conditions exerted an important influence on rabbit microbiota since the relative abundances of many taxa were found differentially represented between both facilities at all taxonomic levels characterized. Furthermore, only five OTUs were needed to achieve a perfect classification of samples according to the facility where animals were raised. The level of feeding and the presence of antibiotics did not modify the global alpha diversity but had an impact on some bacteria relative abundances, albeit in a small number of taxa compared with farm, which is consistent with the lower sample classification power according to these factors achieved using microbial information. CONCLUSIONS: This study reveals that factors associated with the farm effect and other management factors, such as the presence of antibiotics in the diet or the feeding level, modify cecal microbial communities. It highlights the importance of offering a controlled breeding environment that reduces differences in microbial cecal composition that could be responsible for different animal performance.

Bacterial communities from shoreline environments (costa da morte, northwestern Spain) affected by the prestige oil spill.

The bacterial communities in two different shoreline matrices, rocks and sand, from the Costa da Morte, northwestern Spain, were investigated 12 months after being affected by the Prestige oil spill. Culture-based and culture-independent approaches were used to compare the bacterial diversity present in these environments with that at a nonoiled site. A long-term effect of fuel on the microbial communities in the oiled sand and rock was suggested by the higher proportion of alkane and polyaromatic hydrocarbon (PAH) degraders and the differences in denaturing gradient gel electrophoresis patterns compared with those of the reference site. Members of the classes Alphaproteobacteria and Actinobacteria were the prevailing groups of bacteria detected in both matrices, although the sand bacterial community exhibited higher species richness than the rock bacterial community did. Culture-dependent and -independent approaches suggested that the genus Rhodococcus could play a key role in the in situ degradation of the alkane fraction of the Prestige fuel together with other members of the suborder Corynebacterineae. Moreover, other members of this suborder, such as Mycobacterium spp., together with Sphingomonadaceae bacteria (mainly Lutibacterium anuloederans), were related as well to the degradation of the aromatic fraction of the Prestige fuel. The multiapproach methodology applied in the present study allowed us to assess the complexity of autochthonous microbial communities related to the degradation of heavy fuel from the Prestige and to isolate some of their components for a further physiological study. Since several Corynebacterineae members related to the degradation of alkanes and PAHs were frequently detected in this and other supralittoral environments affected by the Prestige oil spill along the northwestern Spanish coast, the addition of mycolic acids to bioremediation amendments is proposed to favor the presence of these degraders in long-term fuel pollution-affected areas with similar characteristics.

Microbial populations related to PAH biodegradation in an aged biostimulated creosote-contaminated soil.

A previous bioremediation survey on a creosote-contaminated soil showed that aeration and optimal humidity promoted depletion of three-ringed polycyclic aromatic hydrocarbons (PAHs), but residual concentrations of four-ringed benzo(a)anthracene (B(a)A) and chrysene (Chry) remained. In order to explain the lack of further degradation of heavier PAHs such as four-ringed PAHs and to analyze the microbial population responsible for PAH biodegradation, a chemical and microbial molecular approach was used. Using a slurry incubation strategy, soil in liquid mineral medium with and without additional B(a)A and Chry was found to contain a powerful PAH-degrading microbial community that eliminated 89% and 53% of the added B(a)A and Chry, respectively. It is hypothesized that the lack of PAH bioavailability hampered their further biodegradation in the unspiked soil. According to the results of the culture-dependent and independent techniques Mycobacterium parmense, Pseudomonas mexicana, and Sphingobacterials group could control B(a)A and Chry degradation in combination with several microorganisms with secondary metabolic activity.

Effects of partially saturated conditions on the metabolically active microbiome and on nitrogen removal in vertical subsurface flow constructed wetlands.

Nitrogen dynamics and its association to metabolically active microbial populations were assessed in two vertical subsurface vertical flow (VF) wetlands treating urban wastewater. These VF wetlands were operated in parallel with unsaturated (UVF) and partially saturated (SVF) configurations. The SVF wetland exhibited almost 2-fold higher total nitrogen removal rate (5 g TN m-2 d-1) in relation to the UVF wetland (3 g TN m-2 d-1), as well as a low NOx-N accumulation (1 mg L-1 vs. 26 mg L-1 in SVF and UVF wetland effluents, respectively). After 6 months of operation, ammonia oxidizing prokaryotes (AOP) and nitrite oxidizing bacteria (NOB) displayed an important role in both wetlands. Oxygen availability and ammonia limiting conditions promoted shifts on the metabolically active nitrifying community within 'nitrification aggregates' of wetland biofilms. Ammonia oxidizing archaea (AOA) and Nitrospira spp. overcame ammonia oxidizing bacteria (AOB) in the oxic layers of both wetlands. Microbial quantitative and diversity assessments revealed a positive correlation between Nitrobacter and AOA, whereas Nitrospira resulted negatively correlated with Nitrobacter and AOB populations. The denitrifying gene expression was enhanced mainly in the bottom layer of the SVF wetland, in concomitance with the depletion of NOx-N from wastewater. Functional gene expression of nitrifying and denitrifying populations combined with the active microbiome diversity brought new insights on the microbial nitrogen-cycling occurring within VF wetland biofilms under different operational conditions.

Rabbit Microbiota Changes Throughout the Intestinal Tract.

To gain insight into the importance of carefully selecting the sampling area for intestinal microbiota studies, cecal and fecal microbial communities of Caldes meat rabbit were characterized. The animals involved in the study were divided in two groups according to the feed intake level they received during the fattening period; ad libitum (n = 10) or restricted to 75% of ad libitum intake (n = 11). Cecum and internal hard feces were sampled from sacrificed animals. Assessment of bacterial and archaeal populations was performed by means of Illumina sequencing of 16S rRNA gene amplicons in a MiSeq platform. A total of 596 operational taxonomic units (OTUs) were detected using QIIME software. Taxonomic assignment revealed that microbial diversity was dominated by phyla Firmicutes (76.42%), Tenericutes (7.83%), and Bacteroidetes (7.42%); kingdom Archaea was presented at low percentage (0.61%). No significant differences were detected between sampling origins in microbial diversity or richness assessed using two alpha-diversity indexes: Shannon and the observed number of OTUs. However, the analysis of variance at genus level revealed a higher presence of genera Clostridium, Anaerofustis, Blautia, Akkermansia, rc4-4, and Bacteroides in cecal samples. By contrast, genera Oscillospira and Coprococcus were found to be overrepresented in feces, suggesting that bacterial species of these genera would act as fermenters at the end of feed digestion process. At the lowest taxonomic level, 83 and 97 OTUs in feces and cecum, respectively, were differentially represented. Multivariate statistical assessment revealed that sparse partial least squares discriminant analysis (sPLS-DA) was the best approach for this purpose. Interestingly, the majority of the most discriminative OTUs selected by sPLS-DA were found to be differentially represented between sampling origins in univariate analysis. Our study provides evidence that the choice of intestinal sampling area is relevant due to important differences in some taxa's relative abundance that have been revealed between rabbits' cecal and fecal microbiota. An appropriate sampling intestinal area should be chosen in each microbiota assessment.